Obesity, which often leads to secondary health complications including heart disease, diabetes, stroke, cancer, and early death, has become one of the primary health concerns in the US. Many neurotransmitters and neuromodulators in the CNS participate in the regulation of energy homeostasis. Dopamine is a great interest in this regard because it promotes food intake, but its role in energy homeostasis is complex and it is often considered as part of a CNS reward system. Here we test the general hypothesis that dopaminergic axons innervate the hypothalamic arcuate nucleus and directly modulate food intake and body weight by regulating the activity of two key neuron types here, but in opposite directions. The arcuate nucleus plays a key role in physiological homeostasis, and particularly energy homeostasis. Two key types of arcuate nucleus neurons are the anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP)/neuropeptide Y (NPY) neurons. The studies proposed below are supported by our preliminary data showing strong innervation of POMC and AgRP neurons by dopamine axons, and by a robust dopaminemediated inhibition of POMC neurons, but excitation of neighboring AgRP neurons. The first Aim examines the structural substrates for interaction between dopamine axons and the POMC and AgRP neurons. We test the hypothesis that dopamine axons make direct synaptic connections with POMC or AgRP neurons using confocal scanning laser microscopy and dual immunolabel electron microscopy with antisera against dopamine to identify dopamine axons. To test the hypothesis that dopamine projections to the ARC arise from the ventral tegmental area (VTA) or other dopaminergic populations, we will use a combination of transgenic mice expressing Cre recombinase under control of the tyrosine hydroxylase or dopamine transporter promoter, coupled with focused intracerebral injections of viral vector containing floxed-stop GFP or tdTomato reporter genes to study efferent projections to the arcuate nucleus from dopaminergic cells. We will also employ injection into the arcuate nucleus of a Cre recombinase-dependent Brainbow-type pseudorabies virus; after retrograde axonal transport the PRV normally expresses a red fluorescent reporter in infected cells, but in dopamine cells expressing Cre, changes color to yellow or blue. Aim 2 tests the hypothesis that dopamine exerts opposing actions on POMC and AgRP neurons, inhibiting POMC cells, but exciting AgRP neurons, both actions enhancing food intake. We will employ transgenic mice expressing various reporter genes, and whole cell voltage- and current clamp recording in hypothalamic brain slices. Whole cell recording will allow us to address different mechanisms of dopamine actions on the POMC and AgRP cells. In Aim 3, a final set of experiments employs optogenetics and transgenic mice expressing Cre recombinase in dopamine neurons, coupled with viral vectors expressing floxed-stop channelrhodopsin-2 (ChR2) or ChIEF. Blue light activates a ChR2- or ChIEF-mediated inward current, allowing us to photostimulate selectively the dopamine axons within the arcuate nucleus. This approach will be used in brain slices to test the hypothesis that light-activated release of transmitter from dopamine axons will exert opposite effects on POMC and AgRP neurons. In mouse in vivo experiments, we test the hypothesis that light-activated transmitter release from dopamine axons will enhance food intake. Food intake, body weight, and activity will be monitored during periods of blue light stimulation of the dopamine axons, and during control periods. Together, these experiments will examine in detail with converging structural, tracing, electrophysiological, and behavioral analyses the hypothesis that dopamine axons innervating arcuate POMC and AgRP neurons play a positive role in energy homeostasis. With the health problems associated with the growing levels of obesity in this country, by some estimates reaching 30% of the adult population, and the associated health complications, knowing and understanding the brain cells involved will help to identify novel approaches to reducing the trend toward obesity.